The vascular tree is a complex network of blood vessels designed to maintain, at its outermost subdivisions, a surface area between blood and tissues for the exchange of gases and nutrients and for the drainage of waste products. During the early stages of inflammation, the sensitive mechanisms relating to microvascular perfusion are altered so that vascular integrity is compromised, blood contents leak into tissues, and hemostasis may develop. In the whole organism, severe and abrupt injury to the microcirculation distorts tissue architecture, impedes delivery of oxygen to cells, and causes extensive fluid loss from the vascular compartment, leading to edema, electrolyte imbalance, shock, and other circulatory disorders. The search for and identification of agents that modulate the immediate responses of inflammation may generate drugs with clinical benefit.
Studies have shown that certain peptides can act as agonists to inhibit inflammation, defined by Cotran et al. (Robbins: Pathologic Basis of Disease, (4th ed., 1989), Ed. Robbins, 2:39-86, Philadelphia: Saunders) as the reaction of vascularized living tissue to local injury. Specific antagonists, by design, work one-on-one against substances that promote inflammation, and the efficacy of a single antagonist may be limited if more than one mediator is released during tissue injury. An agonist, a term introduced by Reuse (Br. J. Pharmacol., 3, pp. 129-62 (1948)) to describe a chemical that activates biological events, would be more efficacious than an antagonist if it could suppress convergent processes initiated by more than one inflammatory mediator. The concept of drugs as anti-inflammatory agonists was discussed by Svensjo and Persson in 1985 (Handbook of Inflammation, Ed. Bonta, 5:51-82, Amsterdam: Elsevier) and by Wei and Thomas ("Anti-Inflammatory Peptide Agonists," Annual Review of Pharmacology and Toxicology, 33, pp. 91-108, 1993).
One or two of us have previously described several different types of peptides useful for anti-inflammatory methods. Thus, U.S. Pat. No. 4,682,930 issued Jan. 9, 1996, titled "Anti-Inflammatory Composition and Method with Des-Tyr Dynorphin and Analogues," inventors Wei and Thomas, describe compounds of the dynorphin family as anti-inflammatory agents.
U.S. Pat. No. 5,480,869, issued Jan. 2, 1996, titled "Anti-Inflammatory Peptide Analogues and Treatment to Inhibit Vascular Leakage in Injured Tissues," inventors Wei and Thomas, describe small peptides having a six amino acid core where one of the amino acid moieties is in the D-configuration.
U.S. Pat. No. 5,374,621, issued Dec. 20, 1994, titled "Neurotensin and Method for Inhibiting Vascular Leakage," inventor Wei, describes neurotensin and analogs useful for anti-inflammatory purposes. Neurotensin is a 13-amino acid residue peptide and is related to an 8-residue peptide, named xenopsin. These peptides affect various physiological functions, such as blood flow, digestion and temperature regulation.
Corticotropin-releasing factor (CRF, also called CRH or corticoliberin) was first characterized as a 41-residue peptide isolated from ovine hypothalami by Vale et al. (1981). Subsequently, the sequence of human-CRF was deduced from cDNA studies and shown to be identical to rat-CRF, and then caprine, bovine, porcine, and white sucker fish CRF were characterized. The CRF of hoofed animals show considerable differences from man, but the pig and fish sequences differ from the human/rat sequence by only 2 out of 41 residues.
Peptides with homologous structures to mammalian CRF are found in cells of certain frog skins and in the urophysis of fish. In fact, the structure of sauvagine, the 40 amino acid peptide isolated from the skins of Phyllomedusa frogs, was reported several years before Vale's description of ovine-CRF in 1981. The structure of sucker fish urotensin I was reported just months after the description of ovine-CRF and resulted from an independent line of inquiry by Lederis's group in Canada. Although sauvagine and urotensin I release adrenocorticotropin (ACTH) from the pituitary, the natural physiological functions of these peptides in the tree-frog (Phyllomedusa species that live in arid regions of South America) and in the sucker fish remain unknown. The mystery deepened when Lederis' group (Okawara et al., "Cloning and Sequence Analysis of cDNA for Corticotropin-Releasing Factor Precursor from the Teleost Fish Catostomus commersoni, " Proc. Natl. Acad. Sci., 85:22, pp. 8439-43 (1988)) showed that the sucker fish not only had urotensin I in its tail-organ but also had CRF in its brain. The co-existence of CRF and urotensin I in the sucker fish suggested that there were other CRF-like peptides in mammals. Vaughan et al., "Urocortin, a Mammalian Neuropeptide Related to Fish Urotensin I and to Corticotropin-Releasing Factor," Nature, 378:6554, pp. 287-92 (Nov. 1995), using antibodies to urotensin I as an investigative tool, recently discovered a CRF-like peptide in rat brain which was called urocortin. The functional inter-relationships and amino acid sequences of CRF superfamily peptides which include CRF, urotensin I-like peptides, and sauvagine are known to the art.
Receptor proteins for CRF were first cloned in 1993 and shown to belong to the second family of 7-transmembrane domain G.sub.s -protein coupled receptors. Two different genes were described encoding the receptors, CRF-R1 and CRF-R2, there being two different splice variants, .alpha. and .beta., for CRF-R2. The distribution of messenger RNA (mRNA) that codes for the synthesis of CRF-R1 is predominantly located in the pituitary, cerebellum, cerebral cortex and olfactory bulb and corresponds well with the location of immunoreactive CRF materials in the brain, as well as CRF binding sites as measured by radio-iodinated ligands. These results indicated that CRF-R1 was the receptor for endogenous CRF and moreover, this relationship was coupled to ACTH release. CRF-R2, however, was found to have a distribution that had little correspondence to the known sites of CRF synthesis. For example, CRF-R2 is found the heart muscle, lung and arterioles of peripheral tissues, but mRNA for CRF synthesis is not found in such tissues.
The pharmacological profile (or pattern of responsiveness) of CRF-R2 receptors to activation by peptides of the CRF superfamily was also clearly different from CRF-R1. Cells transfected with CRF-R1 and CRF-R2 are both sensitive to the cAMP stimulatory effects of human/rat CRF, frog sauvagine, sucker fish urotensin I and rat urocortin. However, the rank order of potency for cAMP stimulation in cells expressing the CRF-R2 receptor was sauvagine&gt;urotensin I=urocortin &gt;h/rCRF whereas sauvagine, urotensin I, urocortin and h/rCRF were about equipotent in cells transfected with CRF-R1 receptor. In assays for ACTH-release, sauvagine and urotensin I are about equipotent to h/rCRF, so again, based on pharmacological profile, it is clear that CRF-R1 is coupled to ACTH-release in physiological systems. The functional activities coupled to CRF-R2 activation are less clear, although it would appear that the natural hormone for activating this system is an urocortin or an urotensin I-like peptide, instead of a CRF-like peptide. In an earlier study (Wei and Kiang, "Peptides of the Corticoliberin Superfamily Inhibit Thermal and Neurogenic Inflammation," European Journal of Pharmacology, 168, pp. 81-86, 1989), it was shown that the intravenous potencies (measured as the median effective dose or ED50) of sauvagine, urotensin I and h/r CRF for suppression of heat-induced edema in the rat were 0.44, 1.5, and 5.9 nmol/kg, respectively. This pharmacological profile, when linked with the fact that CRF-R2 are located in peripheral tissues, indicated that selective activity of agonists at the CRF-R2 receptor paralleled anti-edema potency.
In summary, CRF regulates ACTH secretion via CRF-R1 receptors located on the anterior pituitary. CRF also has several direct actions on central and peripheral tissues, for example CRF has anti-inflammatory inflammatory properties and may have beneficial effects in Alzheimer's disease because it enhances learning and memory in animal models. In these actions on inflammation and memory, CRF is acting mainly on CRF-R2. The natural ligand for CRF-R2 may be peptides such as urocortin, which resemble more closely urotensin I, the peptide that was first identified in fish.
Some therapeutic methods and uses for CRF are described by U.S. Pat. No. 4,801,612, inventors Wei and Kiang, issued Jan. 31, 1989, titled "Method of Inhibiting Inflammatory Response," and U.S. Pat. No. 5,306,710, issued Apr. 26, 1994, titled "Method for Treating Endotoxin Shock with CRF," inventor Wei, which describes use of a CRF to decrease the leakage of blood components into brain tissue produced by various adverse medical conditions, and thus to treat a patient for injury to or disease of the brain, central nervous system or musculature in which edema is a factor.
However, the anti-edema activity of CRF is also associated with its ACTH-releasing activity. This is disadvantageous therapeutically because excessive steroid release can lead to adverse effects, a constellation of symptoms and signs called "Cushing's Syndrome," exhibiting effects such as a loss of muscle mass, thinning of bone, redistribution of fat, etc. Accordingly, a compound for therapeutic use with a more selective anti-edema property but having a reduced, or disassociated, ACTH release would be therapeutically beneficial.
Another limiting factor in the actions of CRF is its binding to CRF binding protein. This protein present in the body, especially in the brain, complexes to CRF with high affinity and reduces the available amount of "free" CRF for pharmacological actions. One approach to reduce CRF's ability to bind to the binding protein has been to modify the human/rat CRF to more closely resemble the ovine variant of CRF. A group of researchers has described some ligand requirements for CRF binding protein in Endocrinology, 136:3, pp. 1097-1102 (Sutton et al., 1995). They showed that in the regions where human/rat CRF differ from ovine CRF, namely, residues 22, 23, and 24 in the 41-amino acid peptide, conversion of the human residues, Ala, Arg and Glu to Thr, Lys, and Asp removes affinity of the variant for the binding protein. Thus, in the synthesis of new analogs, the changes in these three residues will increase the amount of "free" CRSF for pharmacological activities.
Another approach taken in attempts to modify the CRF peptide has been to shorten its overall length. Thus, elimination of resides 1-4 at the N-terminus of ovine CRF has been shown to not alter biological activities or ACTH-release potency. (See Kornreich, J. Med. Chem., 35, pp. 1870-1876 (1992).) Although chemical manipulation of the CRF molecule has modest success in modifying the affinity of CRF for binding protein, and a modest shortening of its length whilst retaining activity, as well as increasing its potency for ACTH-release, so far the features of the CRF that determine receptor selectivity have not been identified. However, a CRF-like compound would be therapeutically useful if it had an anti-edema property but also had reduced, or disassociated, ACTH release.